Most populations of differentiated cells in vertebrates are subject to turnover through cell death and renewal. Some fully differentiated cells such as hepatocytes in the liver and endothelial cells lining the blood vessels simply divide to produce daughter cells of the same differentiated type. The proliferation rate of such cells is controlled to maintain the total number of cells. Thus if a large part of the liver is destroyed then the remaining hepatocytes increase their division rate in order to restore the loss.
Endothelial cells form a single cell layer that lines all blood vessels and regulates exchanges between the blood stream and the surrounding tissues. New blood vessels develop from the walls of existing small vessels by the outgrowth of these endothelial cells which have the capacity to form hollow capillary tubes even when isolated in culture. In vivo, damaged tissues and some tumors attract a blood supply by secreting factors that stimulate nearby endothelial cells to construct new capillary sprouts. Tumors that fail to attract a blood supply are severely limited in their growth.
The process whereby new vessels originate as capillaries, which sprout from existing small vessels, is called angiogenesis. It can therefore be seen that angiogenesis plays a major role in normal tissue development and repair and in the progression of some pathological conditions.
Once the vascular system is fully developed, endothelial cells of blood vessels normally remain quiescent with no new vessel formation. If disease or injury occurs, the formation of new blood vessels can proceed normally, as in natural wound healing, or be insufficient, as in chronic dermal ulcers, or there is deregulation of growth and an abnormal increase in vessel density ensues as in tumorogenesis, diabetic retinopathy, psoriasis and inflammation. Inhibition of inappropriate angiogenesis or enhancement of angiogenesis in non-healing wounds is therefore an extremely important target for drug discovery programs. However, research in this area leading to new drug development has been hindered by the lack of in vitro models of angiogenesis.
Angiogenesis is an extremely complex process involving a wide range of growth factors, extracellular matrix molecules, enzymes and various cell types. Such a complexity of relationships has resulted in major difficulties in developing an in vitro assay which models the entire in vivo process. Angiogenesis can be subdivided into three phases: proliferation, migration and differentiation. Assays exist which model each of these phases separately. Simple in vitro tests measure changes in proliferation of a range of cell types and assess migration over basement membrane proteins. Current in vitro assay systems, which depend on provision of a protein matrix, effectively measure the ability of endothelial cells to differentiate. Assay systems measuring differentiation involve the formation of cord-like structures by endothelial cells. All such systems depend on supplying the cells with exogenous basement membrane proteins on which the cells migrate to form tubules. Cell migration occurs over relatively short time periods of 2-16 hours to give a three dimensional structure. In addition to the basement membrane proteins, many of the systems require the provision of growth factors to produce acceptable tubule formation. The time scale over which tubules are formed provides an excellent test for inhibition of differentiation but is not so useful when testing for enhancement.
The assay systems described above come closest to modeling angiogenesis but none of them combine all three of the stages required for angiogenesis.